The present invention relates to the generation of specific binding partners binding to (poly)peptides encoded by genomic DNA fragments or ESTs. The (poly)peptides are expressed as part of fusion proteins which are forming inclusion bodies on expression in host cells. The inclusion bodies are used to generate binding partners which bind specifically to said (poly)peptides. The specific binding partners, in particular immunoglobulins or fragments thereof, are useful for analysis and functional characterisation of proteins encoded by nucleic acid sequences comprising the corresponding genomic DNA fragments or ESTs.
The invention further relates to nucleic acid molecules, vectors and host cells to be used in the methods of the present invention.
The invention further relates to the use of fusion proteins comprising the first N-terminal domain of the geneIII protein of filamentous phage as fusion partner for the expression of a (poly)peptide/protein fused to said fusion partner, and to methods for the expression of (poly)peptide/proteins.
Since several years, massive efforts are being undertaken to sequence the human genome, and to identify and characterise structure and function of the proteins encoded therein. Finally, this will lead to novel targets for prevention, diagnosis and therapy of diseases (Collins and Galas, 1993; Adams et al., 1995). Currently, two different approaches are being pursued for identifying and characterising the genes distributed along the human genome. In one approach, large fragments of genomic DNA are isolated, cloned, and sequenced. Potential open reading frames in these genomic sequences are identified using bioinformatics software. However, this approach entails sequencing large stretches of human DNA which do not encode proteins in order to find the protein encoding sequences scattered throughout the genome. In addition to requiring extensive sequencing, the bioinformatics software may mischaracterize the genomic sequences obtained. Thus, the software may produce false positives in which non-coding DNA is mischaracterised as coding DNA or false negatives in which coding DNA is mislabelled as non-coding DNA.
In an alternative approach, complementary DNAs (cDNAs) are synthesised from isolated messenger RNAs (mRNAs) which encode human proteins. Using this approach, sequencing is only performed on DNA which is derived from protein coding sequences of the genome. Often, only short stretches of the cDNAs are sequenced to obtain sequences called expressed sequence tags (ESTs) (WO93/00353). In principle, the ESTs may then be used to isolate or purify extended cDNAs which include sequences adjacent to the EST sequences. These extended cDNAs may contain portions or the full coding sequence of the gene from which the EST was derived.
By analysing the genomic DNA or fragments thereof, ESTs, extended cDNAs, and/or the (poly)peptides/proteins encoded thereby, in certain cases, where homology, structural motifs etc. can be identified, it may be possible to assign a function to the (poly)peptide/protein which can be tested or verified in vitro or in vivo. However, the various EST-sequencing efforts have led to enormous numbers of ESTs, and to the problem how best to structure that information and how to identify interesting sequences. Hence, there is still a need for developing and using research tools directed against the (poly)peptide/protein of interest to analyse their localisation on cell and tissue types, their up- or down-regulation in certain disease or development stages or their role in activating or blocking certain interactions or signalling routes. One approach is to use antibodies or fragments thereof as such research tools. In WO93/00353 it was suggested to express the ESTs and to generate antibodies by immunising animals with the corresponding (poly)peptides. In a similar approach, DNA constructs comprising EST sequences have been injected into animals to generate an immune response against the (poly)peptide expressed in vivo (Sykes and Johnston, 1999). However, these approaches are not amenable to a high-throughput generation of antibodies. Alternatively, antibodies are generated against sets of overlapping peptides covering the EST sequence (Persic et al., 1999). In combination with screening recombinant antibody libraries, this approach can in principle be developed to generate antibody fragments as research tools with high throughput. However, it is often difficult to obtain anti-peptide antibodies with sufficiently high affinities.
Thus the technical problem underlying the present invention is to provide a generally applicable method for the generation of specific binding partners binding to (poly)peptides encoded by genomic DNA fragments or by ESTs, especially of antibodies or antibody fragments, for analysis aid functional characterisation of proteins corresponding to genomic DNA or ESTs. The solution to the above technical problem is achieved by providing the embodiments characterised in the claims. The technical approach of the present invention, to provide (poly)peptides encoded by genomic DNA fragments or ESTs for the generation of specific binding partners, such as antibodies or antibody-derived products, by expressing the (poly)peptides as fusions with (poly)peptide/protein fusion partners which lead to the formation of inclusion bodies on expression in host cells, such as E. coli, and to generate specific binding partners against the inclusion bodies and fusion proteins, obtainable therefrom, is neither provided nor suggested by the prior art.
A further problem related to the present invention was to devise a method for the expression of (poly)peptide/proteins which are not easily expressed in free form, e.g. since they are toxic to the host cell. The solution to that technical problem is also achieved by providing the embodiments characterised in the claims. The technical approach of the present invention, express the (poly)peptide/proteins as fusion proteins comprising the first N-terminal domain of the geneIII protein of filamentous phage leading to the formation of inclusion bodies, is neither provided nor suggested by the prior art.
Thus, the present invention relates to a method for generating a specific binding partner to a (poly)peptide which is encoded by a nucleic acid sequence comprised in a genomic DNA fragment or an expressed sequence tag (EST) comprising:
a) expressing a nucleic acid molecule encoding a fusion protein in a host cell under conditions that allow the formation of inclusion bodies comprising said fusion protein, wherein said fusion protein comprises
aa) a (poly)peptide/protein fusion partner which is deposited in inclusion bodies when expressed in said host cell under said conditions and
ab) said (poly)peptide;
b) isolating said inclusion bodies; and
c) generating a specific binding partner that binds specifically to said (poly)peptide.
In the context of the present invention, a xe2x80x9cspecific binding partnerxe2x80x9d is a molecule which is able to specifically bind to a (poly)peptide of interest. Such a specific binding partner may be a peptide, a constrained peptide, an immunoglobulin or fragment thereof, or a cognate binding partner of a naturally occurring protein, e.g. a ligand to a receptor which comprises the (poly)peptide of interest. Such cognate ligand may be obtainable by screening a cDNA expression library for binding to the fusion protein of the present invention. The specific binding partner may also be a non-proteinaceous specific binding partner such as a small molecule, e.g. obtainable by screening of a combinatorial library of small molecules. A specific binding partner may further be modified to enable the detection of an interaction of a specific binding partner and the corresponding (poly)peptide. Such modification may be a detection and/or purification tag (Hochuli et al., 1988; Lindner et al., 1992; Hopp et al., 1988; Prickett et al., 1989; Knappik and Plxc3xcckthun, 1994), or an enzyme (Blake et al., 1984) or a reporter molecule fused or coupled to the specific binding partner. In the context of the present invention, the term xe2x80x9c(poly)peptidexe2x80x9d relates to molecules consisting of one or more chains of multiple, i.e. two or more, amino acids linked via peptide bonds. The term xe2x80x9cproteinxe2x80x9d refers to (poly)peptides where at least part of the (poly)peptide has or is able to acquire a defined three-dimensional arrangement by forming secondary, tertiary, or quaternary structures within and/or between its (poly)peptide chain(s). This definition comprises proteins such as naturally occurring or at least partially artificial proteins, as well as fragments or domains of whole proteins, as long as these fragments or domains have a defined three-dimensional arrangement as described above. The term xe2x80x9cgenomic DNA fragmentxe2x80x9d refers to a contiguous nucleic acid sequence forming part of the genome of an organism and being obtained or obtainable therefrom. The term xe2x80x9cexpressed sequence tags (ESTs)xe2x80x9d are contiguous DNA sequences obtained by sequencing stretches of cDNAs. According to the present invention, such a genomic DNA fragment or EST comprises a nucleic acid sequence which encodes a (poly)peptide or consists of a putative open reading frame (ORF). The EST databases (Eckmann et al., 1998; Bouck et al., 1999) often contain sequences of low sequence quality (Aaronson et al., 1996). One of ordinary skill in the art will be able to identify at least one putative ORFs in a given genomic DNA fragment or EST sequence, and it will not constitute an undue burden for the person skilled in the art to clone all ORFs identified in that way for the expression of a corresponding set of said fusion proteins, and to use them according to the present invention. The length of the genomic DNA fragment or EST is preferably between 100 and 2000 base pairs, more preferably between 200 and 1500 base pairs. The nucleic acid molecule encoding a fusion protein used according to the present invention, or an appropriate vector comprising said nucleic acid molecule, further comprises non-coding DNA sequences which are required to cause or allow the expression of the fusion protein. Methods for construction of nucleic acid molecules encoding a fusion protein used according to the present invention, for construction of vectors comprising said nucleic acid molecules, for introduction of said vectors into appropriately chosen host cells, for causing or achieving the expression of said fusion proteins are well-known in the art (see, e.g., Sambrook et al., 1989; Ausubel et al., 1994). The formation of inclusion bodies can be observed in several host systems in the course of the expression of a (poly)peptide/protein. Inclusion bodies are insoluble aggregates of (poly)peptide/protein deposited within a host cell. They are very dense particles which exhibit an amorphous or paracrystalline structure independent of their subcellular location. Under appropriate conditions the recombinant (poly)peptide/protein deposited in inclusion bodies amounts to about 50% or more of the total cell protein. The formation of inclusion bodies, and their properties, and applications thereof have been investigated in detail (see, for example, Rudolph, 1996; Rudolph and Lilie, 1996; Rudolph et al., 1997; Lilie et al., 1998). Methods of purifying inclusion bodies have been described therein as well and are well-known to one of ordinary skill in the art. The use of inclusion body formation formed by expression of fusion proteins comprising a fusion partner and a (poly)peptide/protein as a general means of expressing said (poly)peptide/protein has been described (WO 98/30684). A fusion partner suitable for a method according to the present invention may be any (poly)peptide/protein which can be found in inclusion bodies when expression in a host cell. In most cases, inclusion body formation is a consequence of high expression rates, regardless of the system or protein used. There seems to be no correlation between the propensity of inclusion body formation of a certain protein and its intrinsic properties, such as molecular weight, hydrophobicity, folding pathways, and so on. (Poly)peptides/proteins where inclusion body formation has been observed and which, therefore, are suitable candidates to be used as fusion partners according to the present invention, include, but are not limited to, E. coli proteins such as maltose-binding protein (Betton and Hofnung, 1996), RNAse II (Coburn and Mackie, 1996), alkaline phosphatase (Derman and Beckwith, 1995), phosholipase A (Dekker et al., 1995), xcex2-lactamase (Rinas and Bailey, 1993), thioredoxin (Hoog, et al., 1984; WO 98/30684), and non E. coli proteins such as human procathepsin B (Kuhelj et al., 1995), porcine interferon-xcex3 (Vandenbroeck et al., 1993), or T5 DNA polymerase (Chatterjee et al., 1991). The host referred to above may be any of a number commonly used in the production of proteins, including but not limited to bacteria, such as E. coli (see. e.g., Ge et al, 1995) or Bacillus subtilis (Wu et al., 1993); fungi, such as yeasts (Horwitz et al., 1988; Ridder et al., 1995) or filamentous fungus (Nyyssxc3x6nen et al., 1993); plant cells (Hiatt, 1990, Hiatt and Ma, 1993; Whitelam et al., 1994), insect cells (Potter et al., 1993; Ward et al., 1995), or mammalian cells (Trill et al., 1995).
The generation, and optionally, identification, of xe2x80x9ca binding partner that binds specifically to said (poly)peptidexe2x80x9d can be achieved by using a variety of methods, depending on the type of specific binding partner, which are well-known to one of ordinary skill in the art. For example, combinatorial libraries of chemical compounds, peptides or biomolecules, such as immunoglobulins, can be screened and/or selected against the isolated inclusion body as target, preferably after purification, or, more preferably, against the fusion protein obtained from said inclusion bodies, either in solubilised or in refolded form, or against the free (poly)peptide as target (see, for example: Pinilla et al., 1999; Woodbury and Venton, 1999; Borman, 1999; Eisele et al., 1999; Lebl, 1999).
In a preferred embodiment of the method of the invention, said fusion protein comprises said fusion partner as N-terminal portion and said (poly)peptide as C-terminal portion. Further preferred is a method, wherein said fusion protein further comprises a (poly)peptide linker linking said fusion partner and said (poly)peptide. The linker may consist of about 1 to about 30, preferably of between about 5 and about 15 amino acids. Particularly preferred is a method, wherein, said linker comprises a cleavage signal. In the context of the present invention, the term xe2x80x9ccleavage signalxe2x80x9d refers to a amino acid sequences which allows to cleave, e.g. by chemical or enzymatic reactions, the fusion protein between said fusion partner and said (poly)peptide to be able to obtain said (poly)peptide in free form. Such cleavage signal is preferably a specific recognition sequence of a protease well known to one of ordinary skill in the art, such as enterokinase or thrombin. Alternatively, the fusion protein might be cleaved by chemical cleavage with a chemical such as cyanogen bromide.
Said fusion protein may further comprise additional (poly)peptide sequences at N- and/or C-terminus, and/or in said (poly)peptide linker. This comprises, for example, (poly)peptides which allow to identify and/or purify said fusion protein. Examples for such (poly)peptide tags are Hisn (Hochuli et al., 1988; Lindner et al., 1992), myc, FLAG (Hopp et al., 1988; Prickett et al., 1989; Knappik and Plxc3xcckthun, 1994), or a Strep-tag (Schmidt and Skerra, 1993; Schmidt and Skerra, 1994; Schmidt et al., 1996). These tags are all well known in the art and are fully available to the person skilled in the art.
In a yet further preferred embodiment of the method of the invention, said genomic DNA fragment or said EST is obtained from a prokaryotic organism or from a virus. Most preferred is a method wherein said prokaryotic organism or virus is a pathogen.
By sequencing the genome of organisms pathogenic to human, or pathogenic to animals or plants, new proteinaceous targets for prevention, diagnosis and/or therapeutic intervention are being sought.
Further preferred is a method wherein said nucleic acid is expressed under conditions allowing over-expression of said fusion protein.
In a further preferred embodiment, the invention relates to a method wherein said genomic DNA fragment or said EST is obtained from a eukaryotic organism.
In a preferred embodiment, the present invention relates to a method wherein said genomic DNA fragment or said EST is obtained from a non-mammalian species.
Further preferred is a method wherein said genomic DNA fragment or said EST is obtained from a mammalian species.
In a most preferred embodiment the present invention relates to a method wherein said mammalian species is human.
In a preferred embodiment of the method of the invention, said host cell is a eukaryotic cell. Particularly preferred is a yeast or insect cell.
In a most preferred embodiment of the method of the invention, said host cell is a prokaryotic cell. Particularly preferred is a bacterial cell. Most preferably, said bacterial cell is an E. coli cell.
An additional preferred embodiment of the invention relates to a method wherein said fusion protein is expressed in the cytosol of a bacterial host cell. Particularly preferred is the cytosolic expression of fusion proteins according to the present invention wherein said fusion partner contains at least one disulfide bond. It has been found that inclusion body formation can be anticipated if a disulfide bonded (poly)peptide/protein is produced in the bacterial cytosol, as formation of disulfide bonds does usually not occur in this reducing cellular compartment. The consequence is improper folding resulting in aggregation (Lilie et al., 1998).
Further preferred is a method where said fusion partner is a secreted protein, and wherein said nucleic acid does not comprise a nucleic acid sequence encoding a signal sequence for the transport of the fusion protein to the periplasm. It has been observed that cytosolic expression of secreted (poly)peptide/protein leads to the formation of inclusion bodies (Lilie et al., 1998).
In a preferred embodiment the present invention relates to a method wherein said fusion partner is an endogenous (poly)peptide/protein of said host cell.
Most preferred is a method wherein said fusion partner is a (poly)peptide/protein foreign to said host cell. Particularly preferred is a method wherein said fusion partner is taken from the list of E. Coli maltose-binding protein, E. coli RNAse II, E. coli alkaline phosphatase, E. coli phosholipase A, E. coli xcex2-lactamase, E. coli thioredoxin, human procathepsin B, porcine interferon, and T5 DNA polymerase.
In a further most preferred embodiment of the method of the invention, said host cell is E. coli and said fusion partner comprises the first N-terminal domain of the geneIII protein of a filamentous phage. Preferably, said fusion partner consists of the two N-terminal domains of the geneIII protein, more preferably of the first N-terminal domain of the geneIII protein. Most preferably, said fusion partner consists of amino acids 1 to 82 of the geneIII protein.
Infection of Escherichia coli by the Ff filamentous phages fl, fd, and M13 is initiated by interaction of the geneIII protein (g3p) located at one end of the phage particle with the tip of the F conjugative pilus (Model and Russel, 1988). Mature g3p (406 amino acids) consists of 3 domains separated by linker sequences (Stengele et al., 1990; Krebber et al., 1997). The following roles could be assigned to the individual domains: The N-terminal domain of g3p (N1) is responsible for membrane penetration (Riechmann and Holliger, 1997), the middle domain (N2) for binding of the bacterial F-pilus (Stengele et al., 1990) and the C-terminal domain (CT) plays a role in phage morphogenesis and caps one end of the phage particle (Crissman and Smith, 1984). The crystal structure of the two N-terminal domains of g3p (N1-N2) and the solution structure of N1 have been solved (Lubkowski et al., 1998; Holliger and Riechmann, 1997). Purified N1 was shown to be highly soluble and monomeric at mM concentrations (Holliger and Riechmann, 1997). Expression of N1 or N1-N2 in the cytoplasm of E. coli, however, leads to the formation of inclusion bodies from which the proteins can be refolded (C. Krebber, 1996; Krebber et al., 1997). Since expression of N1 and N1-N2fusion proteins are toxic to the cells (C. Krebber, 1996), tight regulation of transcription of the fusion genes are preferred using for example the pET (Stratagene, La Jolla, Calif., USA) or the pBAD expression system (Invitrogen BV, Groningen, The Netherlands). The use of these vectors is in all cases applicable where toxic effects of gene products is being expected, assumed or observed, and is one of the first steps well known to one of ordinary skill in the art in adjusting expression conditions. Fusion partners comprising the first N-terminal domain of guIIIp are particularly useful since the fusion protein readily form inclusion bodies on cytosolic expression, but are easily solubilised (Krebber et al., 1997). The fusion partner may also be a variant or a mutant of a parental fusion partner referred to hereinabove (such as a (poly)peptide/protein comprising the first N-terminal domain of gIIIp), provided that such variant or mutant is deposited in inclusion bodies as well when expressed in host cell under conditions where the parental fusion partner is deposited in inclusion bodies. Such variant or mutant may result from the parental fusion partner e.g. by adding, substituting and/or deleting one or more amino acid residue(s). Since the formation of inclusion bodies on expression is a property which can easily be monitored by one of ordinary skill in the art, it does not require an undue burden of experimentation to identify variants or mutants with properties suitable for the methods of the present invention.
In a further preferred embodiment, the invention relates to a method wherein step b) further comprises the step of (i) solubilising said fusion protein under suitable conditions. In a yet further preferred embodiment, the present invention relates to a method wherein step b) further comprises the step of (ii) refolding said fusion protein under suitable conditions.
Methods for solubilising and/or refolding (poly)peptides/proteins found deposited in inclusion bodies have been thoroughly investigated and are well known to the practitioner of ordinary skill in the art (see, for example, Rudolph, 1996; Rudolph and Lilie, 1996; Rudolph et al., 1997; Lilie et al., 1998).
In another preferred embodiment, the invention relates to a method wherein said fusion protein further comprises a (poly)peptide linker linking said fusion partner and said (poly)peptide, wherein said linker comprises a cleavage signal, and wherein step b) further comprises the steps of (iii) cleaving said fusion protein between said fusion partner and said (poly)peptide, and (iv) isolating said (poly)peptide in free form. Further preferred is a method further comprising the step of purifying said fusion protein or said (poly)peptide in free form.
The construction of fusion proteins comprising a cleavage signal which allows to cleave the fusion protein between said fusion partner and said (poly)peptide has been described hereinabove.
In a preferred embodiment of the method of the invention, said specific binding partner is an immunoglobulin or a fragment thereof
In this context, xe2x80x9cimmunoglobulinxe2x80x9d is used as a synonym for xe2x80x9cantibodyxe2x80x9d. Immunoglobulin fragments according to the present invention may be Fv (Skerra and Plxc3xcckthun, 1988), scFv (Bird et al., 1988; Huston et al., 1988), disulfide-linked Fv (Glockshuber et al., 1992; Brinkmann et al., 1993), Fab, (Fabxe2x80x2)2 fragments or other fragments well-known to the practitioner skilled in the art, which comprise the variable domain of an immunoglobulin or immunoglobulin fragment. Particularly preferred is the scFv fragment format.
In a most preferred embodiment of the method of the invention, said immunoglobulin or fragment thereof is generated by (i) immunisation of an animal with said inclusion bodies, said fusion protein or said (poly)peptide, and (ii) by selecting an immunoglobulin produced by said animal which specifically binds to said inclusion bodies, said fusion protein or said (poly)peptide. Methods for immunising animals and for screening and/or selection of specific immunoglobulin are well-known to one of ordinary skill in the art.
In a further most preferred embodiment of the method of the invention, said immunoglobulin or fragment thereof is generated by selecting a member of a recombinant library of immunoglobulins or fragments thereof which specifically binds to said inclusion bodies, said fusion protein or said (poly)peptide. Recombinant libraries of immunoglobulins or fragments thereof have been described in various publications (see, e.g., Vaughan et al., 1996; Knappik et al., 2000; WO 97/08320), and are well-known to one of ordinary skill in the art.
Particularly preferred is a method wherein said library is displayed on the surface of a replicable genetic package.
The term xe2x80x9creplicable genetic packagexe2x80x9d refers to an entity which combines phenotype and genotype of members of a library of (poly)peptides/proteins by linking the genetic information encoding the library member and the (poly)peptide/protein expressed therefrom. The library can be screened and/or selected for a desired property, and the (poly)peptide/protein being screened and/or selected can be identified via the genetic information associated with the same. Examples for xe2x80x9creplicable genetic packagesxe2x80x9d comprise cells, such as bacteria (WO 90/02809; Georgiou et al., 1993; Francisco and Georgiou, 1994; Daugherty et al., 1998), yeast (Boder and Wittrup, 1997; Kieke et al., 1997; Cho et al., 1998; Kieke et al., 1999) insect cells (Ernst et al., 1998), viruses, such as bacteriophage (WO 90/02809; Kay et al., 1996; Dunn, 1996; McGregor, 1996) retroviruses (Russell et al., 1993), spores (WO 90/02809), or complexes of nucleic acid molecules and (poly)peptides/proteins expressed therefrom, such as in ribosome complexes (Hanes and Plxc3xcckthun, 1997; Hanes et al., 1998; Hanes et al., 1999) or in complexes connected either non-covalently (Cull et al., 1992; Schatz, 1993; Schatz et al., 1996; Gates et al., 1996) or covalently (Nemoto et al., 1997).
Further preferred is a method wherein said replicable genetic package is a filamentous phage.
In the context of the present invention, the term xe2x80x9cfilamentous phagexe2x80x9d refers to a class of bacteriophage which are able to infect a variety of Gram negative bacteria. They have a single-stranded, covalently closed DNA genome which is packaged in a protein coat forming a long cylinder. The best characterised of these phage are M13, fd, and fl and derivatives thereof. Filamentous phage have been used extensively for the display of foreign (poly)peptides/proteins and libraries thereof, and the various approaches and applications have been reviewed in several publications (e.g. Kay et al., 1996; Dunn, 1996; McGregor, 1996).
Particularly preferred is the use of a fusion protein comprising the N-terminal domain of the geneIII protein (g3p) of filamentous phage as fusion partner for biopanning of a recombinant library of immunoglobulins or fragments thereof displayed on the surface of filamentous phage. The following properties of N1 make it an especially suitable candidate to be used in biopanning of phage display libraries:
N1 (amino acids 1-82 of the mature g3p) is small and has a low pI of 4.14, which is advantageous for coating to conventional micro titer plates used for biopanning which is routinely done at physiological pH
most phages displaying N1-binding scFvs on their surface should automatically be removed since they should bind to other phages which carry 3-5 copies of g3p comprising N1 on their surface.
In another embodiment, the present invention relates to a nucleic acid molecule encoding a fusion protein comprising aa) the first N-terminal domain of the geneIII protein of filamentous phage and ab) a (poly)peptide which is encoded by a nucleic acid sequence comprised in a genomic DNA fragment or an expressed sequence tag (EST), wherein said nucleic acid molecule does not comprise a nucleic acid sequence encoding a signal sequence for the transport of the fusion protein to the periplasm of a bacterial host cell.
In a further embodiment, the invention relates to a vector which comprises a nucleic acid molecule of the present invention.
Preferably, said vector is an expression vector.
In another embodiment, the invention relates to a host cell comprising a nucleic acid or a vector according to the present invention.
Particularly preferred is a host cell which is an E. coli cell.
Additionally, the invention relates to the use of a fusion protein comprising the first N-terminal domain of the geneIII protein of filamentous phage as fusion partner for the expression of a (poly)peptide/protein fused to said fusion partner, wherein said fusion protein is obtained in the form of inclusion bodies. The general method of using inclusion body formation formed by expression of fusion proteins comprising a fusion partner and a (poly)peptide/protein as a means of expressing said (poly)peptide/protein has been described (WO 98/30684). The fusion protein may further comprise a linker sequence linking said fusion partner and said (poly)peptide/protein. The linker may consist of about 1 to about 30, preferably of between about 5 and about 15 amino acids. The linker may comprise a cleavage signal which allows to cleave the fusion protein between the fusion partner and the (poly)peptide/protein to be able to obtain said (poly)peptide/protein in free form. Such cleavage signal is preferably a specific recognition sequence of a proteases well known to one of ordinary skill in the art, such as enterokinase or thrombin. Alternatively, the fusion protein might be cleaved by chemical cleavage with a chemical such as cyanogen bromide. Such fusion proteins, after refolding, can be used in in vitro SIP as well (Krebber et al., 1997).
The invention furthermore relates to a method for the expression of a (poly)peptide/protein comprising:
a) expressing a nucleic acid molecule encoding a fusion protein in a host cell under conditions that allow the formation of inclusion bodies comprising said fusion protein, wherein said fusion protein comprises
aa) the first N-terminal domain of the geneIII protein of filamentous phage, and
ab) said (poly)peptide/protein.
Particularly preferred is a method further comprising the steps of
b) isolating said inclusion bodies, and
c) solubilising said fusion protein under suitable conditions.
The specific binding partners generated according to the present invention may be used for the identification and/or characterisation of a naturally occurring (poly)peptide/protein comprising said (poly)peptide. Such uses include, but are not limited to, the use of specific binding partners such as immunoglobulins or fragments thereof in immunoassays such as ELISA, in Western blot analysis of cell extracts, immunohistochemistry or immunocytochemistry on tissues or cells, immunoprecipitations, immunocoprecipitation using cell extracts, and so on. The use of specific binding partners such as immunoglobulins or fragments thereof in such binding assays, or in similar methods, and in the isolation of target material is well-known to one of ordinary skill in the art.
By using the specific binding partner generated according to the present invention it will be possible to identify and/or characterise naturally occurring (poly)peptide/protein comprising said (poly)peptide. Methods for isolating naturally occurring (poly)peptides/proteins from natural sources, and methods for the identification of these (poly)peptide/protein, either directly or via the genetic information encoding these (poly)peptide/protein, are well-known to one of ordinary skill in the art.